This application is a Rule 371 Application of PCT Application No. PCT/NL98/00357 (Publication No. WO 9858678), filed on Jun. 22, 1998 which claims priority to EP 97201895.6, filed on Jun. 20, 1997.
This invention relates to methods of treating diseases of in which the immune system is involved. In particular, this invention relates to methods of treating T-cell mediated autoimmune diseases such as multiple sclerosis and various malignancies of lymphoid origin.
Immunotoxins:
Immunotoxins are chimeric molecules in which cell-binding ligands are coupled to toxins or their subunits. The ligand portion of the immunotoxin is usually a monoclonal antibody (Mab) that binds to selected target cells. The toxin portion of the immunotoxin can be derived form various sources. Most commonly, toxins are derived from plants or bacteria, but toxins of human origin or synthetic toxins (drugs) have been used as well. Toxins used for immunotoxins derived from plants or bacteria all inhibit protein synthesis of eukaryotic cells. The most widely used plant toxin ricin, consist of two disulfate-linked polypeptides A and B (Olsnes et al., in Molecular Action of Toxins and Viruses p51-105 (1982)). The most widely used bacterial toxin is Pseudomonas exotoxin (PE). Pseudomonas exotoxin is produced by the bacterium as a single-chain protein (Allured et al., Proc. Natl. Acad. Sci. USA 83:1320 (1986)). Another group of plant-derived toxins used in ITs are single-chain proteins (type I RIP), frequently found in plants and have similar enzymatic properties as the A-chain of ricin (reviewed in Stirpe and Barbieri FEBS Lett. 195:1 (1986)), these type I RIP however lack the B-chain. The absence of binding activity and as a consequence the inability of the native toxin to bind to cells significantly decreases the non-specific toxicity and makes these toxins extremely interesting for usage in ITs. To target these toxins to potential harmful cells, they are coupled to a Mab against a specific protein on the surface of these targeted cells. The cross-linker used to join the Mab and the toxin must remain stable extracellular, but after internalization of the conjugate into the cell, be labile intracellular so that the toxin fragment can be released in the cytosol and target to the appropriate intracellular location. A complete Mab consists of two heavy and two light chains and can be chemically coupled to the toxin. Using this chemical coupling usually several toxin molecules are coupled to one Mab molecules, resulting in protein complexes of considerable size. An alternative to complete Mabs, is to use single-chain antibody fragments (scFv), which consist of only the variable part of the heavy chain (VH) and the variable part of the light chain (VL) coupled via a short linker (Pastan et al., Annu. Rev. Biochem. 61:331 (1992)). The usage of scFv-ITs has a number of advantages compared to chemically coupled ITs. First, the scFv can be cloned via a short linker to a toxin and can be expressed as fusion-protein in a bacterial expression system. Secondly, tissue penetration is a major obstacle when chemical coupled IT-conjugates were used in various animal models, the scFv format being superior in this respect. The use of a scFv as ligand portion of an immunotoxin reduces the size of ligand portion with a factor 6 as compared to a complete Mab and in these recombinant molecules one toxin molecule is linked to one scFv reducing the size even further. Since a scFv-IT is produced as one molecule unwanted cleavage of the toxin and the ligand in the circulation can not occur. The capability of scFv-ITs to specifically eliminate cells, revealed that intracellular cleavage of the ligand and toxin part, that is necessary when ITs are used, is not necessary for the cytotoxic effect of certain scFv-ITs.
Various types of immunotoxins directed against different cellular targets have been evaluated in vivo, both in animal models and in phase I or II clinical trials. The vast majority of clinical studies with immunotoxins has been performed for anti-tumor therapy using ricin A chain or blocked ricin (Frankel et al., Leukemia and Lymphoma 26:28 (1997), Lynch et al., J. Clin. Onc. 15:723 (1997)). Reports on the administration of immunotoxins containing type I RIPs are limited. Thusfar only two studies have been published, the first using saporin-S6 coupled to an anti-CD30 Mab (Falini et al., Lancet 339:1195 (1992)) and the second using the PAP toxin coupled to an anti-CD19 Mab (Uckun et al., Blood 79:3369 (1992)). An increasing number of preclinical studies using immunotoxins containing various different type I RIPs (momordin, gelonin, saporin, bryodin and bouganin) are currently under development.
The CD40L Molecule:
The CD40L molecule belongs to the TNF/CD40L gene family (Armitage et al., Curr. Opin. Immunol. 6:407 (1994)). Although TNF is a soluble cytokine, it is initially synthesized as a membrane associated molecule. Most of the members of the of the TNF/CD40L receptor family are type II transmembrane proteins. Initially it was reported that the expression of the CD40L was restricted to activated CD4+ T cells. Now it has also been detected on B cells from autoimmuine patients, on mast cells and on baophils. The cell surface expression of CD40L is tightly regulated, specific signals are needed for its appearance and, once engaged with CD40 the molecule rapidly disappears again.
Autoimmune Diseases:
A normal functioning immune system has self-regulating mechanisms to terminate the immune response when it is no longer needed. When these self-regulatory mechanisms become compromised, a person may develop a so-called autoimmune disease. Examples of autoimmune diseases are rheumatoid arthritis, multiple sclerosis, type I diabetes, lupus, thyroiditis, systemic lupus erythematosus and myasthenia gravis.
Multiple sclerosis (MS) is a severely disabling progressive neurological disease, involving autoimmune attack against myelin in the central nervous system. MS affects 1 in 1000 in the USA and Europe. Due to improved diagnosis that number is currently increasing. Onset of disease is usually around 30 years of age and, on average, patients are in need of treatment for another 28 years. Diagnosis of exacerbations and early identification of onset of exacerbations has improved greatly, allowing design of novel treatment strategies. Recently, the involvement of the CD40L molecule in the pathophysiology of MS has been demonstrated using the experimental allergic encephalomyelitis model in mice (Gerritse et al., Proc. Natl. Acad. Sci. USA 93:2499 (1996)). In this model, injection of mice with a blocking monoclonal antibody (Mab) to CD40L at the time of disease induction, completely prevents disease. Furthermore, in situ analysis of CD40L and CD40 in human MS brain has revealed that CD40 expression is abundantly expressed on macrophages in perivascular infiltrates. Frequencies of CD40L positive cells in these infiltrates were modest, but could be found in juxtaposition to CD40 positive cells, indicative of an ongoing cellular interaction.
Systemic lupus erythematosus (SLE) is an other autoimmune disease in which the CD40L molecule has been implicated. SLE, in contrast to most autoimmune diseases, has the potential to involve multiple organs. The clinical manifestations of SLE are extremely variable and diverse. Some patients only have mild involvement of skin and joints, require little medication and show spontaneous remissions. Whereas other patients suffer from severe and progressive glomerulonephritis that in the end does not even respond to high doses steroids and cyclophosphamide. SLE can manifest at nearly any age, but the disease onset is usually between 15 and 50 years. SLE affects about 8 times more females than males. The chance that a caucasian women in her life time develops SLE is about 1 in 700, whereas this incidence can be two to four times higher in hispanics or blacks. The overall prevalence of SLE is in the order 1 in 2000. SLE is characterized by a production of high affinity IgG antibodies to self antigens (autoantibodies). The principal targets of autoantibodies in SLE include certain protein-nucleic acid complexes. The multivalent nature of these complexes and their ability to cross-link B-cell receptors have been proposed as explanations for their strong immunogenicity. However, the mechanism by which these autoantibodies cause disease is still unclear. Autoantibodies to phospholipids are also frequently found and are associated with thrombotic complications. Also autoantibodies to cell surface molecules can be found. These target specificities are easier to understand with respect to the pathology, causing problems such as hermolytic anemia and platelet destruction. In contrast to autoimmune diseases such as RA and MS, T cells do not appear to play a direct role in tissue damage in SLE, although the do play an important role in the production of autoantibodies. The induction of the CD80/86 molecules on autoantigen-specific B cells by autoantigen-specific helper T cells via the CD40L-CD40 interaction represents a critical step in the maturation and subsequent differentiation of autoantigen-specific B cells. Recently it was observed that both T cells and also B cells from active SLE patients show a constitutive expression of CD40L (Desai-Metha et al., J. Clin. Invest. 97:2063 (1996)).
Activated T cells are specifically involved in the pathophysiology of autoimmune diseases such as MS and SLE. The onset of an exacerbation in autoimmune patients is believed to started when autoreactive T cells are activated. This antigen-specific activation results in the express significant amounts of CD40L on the cell surface. This has led several groups to explore the physical blocking of the CD40L-CD40 interaction as a treatment modality for auto immune diseases. However, it may be expected that after withdrawal of the therapeutic molecule that blocks the CD40L-CD40 interaction, the disease can return to the same magnitude or even more severe as before the treatment. As an alternative, it is being explored to selectively inactivate autoantigen-specific T cells with modified autoantigens. However, it has not previously been proposed to use the CD40L receptor on activated T cells to selectively eliminate autoantigen-specific T cells from circulation of autoimmune patients. The present inventors propose a selective method for the treatment of autoimmuine diseases such as MS and SLE, that is based on the selective killing of the CD40L-positive autoantigen-specific T cells by an anti-CD40L immunotoxin fusion protein. The significant advantage of the use of an anti-CD40L immunotoxin fusion protein over the use of blocking anti-CD40L Mabs is that after several rounds of anti-CD40L immunotoxin treatment, all autoreactive T cells will have been deleted from the patients T-cell repertoire possibly resulting in a cure from the disease.
The current invention is thus based on the discovery that conjugates of antibodies to human CD40L and a toxin (immunotoxin) can effectively kill cells expressing the CD40L molecule. Accordingly, these anti-CD40L immunotoxins can be used to prevent or treat diseases or conditions that are mediated by the cells expressing the CD40L molecules. Accordingly, it is a primary object of this invention to provide an immunotoxin comprising a Mab capable of binding to the human CD40L antigen located on the surface of activated human lymphocytes and a toxin molecule, wherein the binding of said immunotoxin to the CD40L positive cell results in cell death.
It is another object of this invention to provide a method for treating autoimmune diseases such as multiple sclerosis, psoriasis, rheumatoid arthritis and systemic lupus erythematosus in a patient, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of an immunotoxin capable of binding to the human CD40L antigen located an the surface of activated human lymphocytes, wherein the binding of said immunotoxin to the CD40L antigen results in elimination of the CD40L expressing cells and inhibition of the local inflammatory response, in a pharmaceutically acceptable excipient.
It is a further object of this invention to provide a method for treating malignancies of lymphoid origin in a patient, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of an IT capable of binding to the human CD40L antigen located on the surface of malignant cells of lymphoid origin, wherein the binding of the IT to the CD40L antigen results in elimination of the CD40L expressing tumor cells, in a pharmaceutically acceptable excipient.
The invention described herein draws on previously published work and pending patent applications. By way of example, such work consists of scientific papers, patents or pending patent applications. All of these publications and applications, cited previously or below are hereby incorporated by reference.
As used herein, the term xe2x80x9cimmunotoxinxe2x80x9d refers to chimeric molecules in which a cell binding ligand is coupled a toxin or its sub-unit. The toxin portion of the immunotoxin can be derived from various sources, such as plants or bacteria, but toxins of human origin or synthetic toxins (drugs) can be used as well.
Preferably, the toxin part is derived form a plant toxin, such as a ribosome inactivating protein (RIP), type-1 or type-2. A type-2 RIP includes for example ricin (see Olsnes et al, 1982, above). Type-1 RIP""s are particularly useful for constructing immunotoxins according to the invention. Type-1 RIP""s include pokeweed anti-viral protein (PAP) (reviewed by Irvin, in Antiviral Proteins in Higher Plants 65 (1994)), bryodin (see e.g. EP-A-710723, U.S. Pat. No. 5,597,569 and EP-A-725823, GB-A-2194948), momordin (see WO97/19957, WO92/14491, JP-A-51-67714), gelonin (see e.g. WO94/26910, WO93/09130, WO93/20848, WO93/05168), saporin (see Falini et al, 1992, above), and, bouganin (see co-pending patent application PCT/NL98/00336). An example of bacterial toxins is Pseudomonas exotoxin (see EP-A-583794, Allured et al, 1986 above).
The term xe2x80x9cligandxe2x80x9d may refer to all molecules capable of binding with or otherwise recognizing a receptor on a target cell. Examples of such ligands include, but are not limited to, antibodies, growth factors, cytokines, hormones and the like, that specifically bind desired target cells.
As used herein, the term xe2x80x9cantibodyxe2x80x9d refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as Fab, F(abxe2x80x2)2, Fv, and other fragments which retain the antigen binding function of the parent antibody.
As used herein, the term xe2x80x9cmonoclonal antibodyxe2x80x9d refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins as well as fragments such as Fab, F(abxe2x80x2)2, Fv, and others which retain the antigen binding function of the antibody. Monoclonal antibodies of any mammalian species can be used in this invention. In practice, however, the antibodies will typically be of rat or murine origin because of the availability of rat or murine cell lines for use in making the required hybrid cell lines or hybridomas to produce monoclonal antibodies.
As used herein, the term xe2x80x9chumanized antibodiesxe2x80x9d means that at least a portion of the framework regions of an immunoglobulin are derived from human immunoglobulin sequences.
As used herein, the term xe2x80x9csingle chain antibodiesxe2x80x9d refer to antibodies prepared by determining the binding domains (both heavy and light chains) of a binding antibody, and supplying a linking moiety which permits preservation of the binding function. This forms, in essence, a radically abbreviated antibody, having only that part of the variable domain necessary for binding to the antigen. Determination and construction of single chain antibodies are described in U.S. Pat. No. 4,946,778 to Ladner et al., Methods for the generation of antibodies suitable for use in the present invention are well known to those skilled in the art and can be found described in such publications as Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).
The immunotoxin molecules of the present invention may be generated by conjugation of a CD40L binding ligand to a toxin by any method known and available to those skilled in the art. The ligand and the toxin molecules may be chemically bonded together by any of a variety of well-known chemical procedures, such as the use of heterobifunctional cross-linkers, e.g. SPDP, carbodiimide or glutaraldehyde. Production of various immunotoxins is well-known within the art and can be found, for example in xe2x80x9cMonoclonal Antibody-Toxin Conjugates: Aiming the Magic Bulletxe2x80x9d, Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp.168-190 (1982) and Waldmann, Science, 252:1657 (1991), both of which are incorporated by reference.
The ligand may also be fused to the toxin by recombinant means such as through the production of single chain antibody-toxin fusion proteins. The genes encoding ligand and the toxin may be cloned in cDNA form and linked directly or separated by a small peptide linker by any cloning procedure known to those skilled in the art. See for example Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, (1989).
A person skilled in the art will realize that additional modifications, deletions and insertions may be made to the ligand binding agent and the toxin genes. Especially, deletions or changes may be made in the linker connecting a ligand gene to the toxin. All such constructions may be made by methods of genetic engineering well known to those skilled in the art (see, generally, Sambrook et al., supra) and may produce proteins that have differing properties of affinity, specificity, stability and toxicity that make them particularly suitable for various clinical or biological applications.
The pharmaceutical compositions of this invention are administered at a concentration that is therapeutically effective to a patient in the need of a treatment. To accomplish this goal, the pharmaceutical compositions may be formulated using a variety of acceptable excipients known in the art. The compositions for administration will commonly comprise a solution of the anti-CD40L immunotoxin molecule dissolved in a pharmaceutical acceptable carrier, preferably an aqueous carrier. Typically, the immunotoxins are administered by injection, either intravenously, intraperitoneally, in an other body cavity or or into a lumen of an organ. Methods to accomplish this administration are known to those of ordinary skill in the art. It may also be possible to obtain compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes.
Before administration to patients, formulants may be added to the antibodies. A liquid formulation is preferred. For example, these formulants may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Preferably carbohydrates include sugar or sugar alcohols such as mono-, di-, or polysaccharides. The saccharides can include fructose, glucose, mannose, sorbose, xylose, lactose, maltose, sucrose, dextan, pullulan, dextrin, xcex1- and xcex2-cyclodextrin, soluble starch, hydroxyethyl starch, carboxymethyl cellulose, other water-soluble glucans, or mixtures thereof. Sucrose is most preferred. xe2x80x9cSugar alcoholxe2x80x9d is defined as a C4 to C8 hydrocarbon having an xe2x80x94OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is most preferred. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. Preferably, the sugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %, more preferable between 2.0 and 6.0 w/v %. Preferably amino acids include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added. Preferred polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. It is also preferred to use a buffer in the composition to minimize pH changes in the solution before lyophilization or after reconstitution. Most any physiological buffer may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are preferred. Most preferred is a citrate buffer. Preferably, the concentration is from 0.01 to 0.3 molar. Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268,110.
Additionally, antibodies can be chemically modified by covalent conjugation to a polymer to increase their circulating half-life, for example. Preferred polymers, and methods to attach them to peptides, are shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546 which are all hereby incorporated by reference in their entireties. Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula: R(Oxe2x80x94CH2xe2x80x94CH2)nOxe2x80x94R where R can be hydrogen, or a protective group such as an alkyl or alkanol group. Preferably, the protective group has between 1 and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. The PEG has a preferred average molecular weight between 1000 and 40,000, more preferably between 2000 and 20,000, most preferably between 3,000 and 12,000. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this hydroxy group which is preferably activated to react with a free amino group on the inhibitor. However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/antibody of the present invention.
Water soluble polyoxyethylated polyols are also useful in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), etc. POG is preferred. One reason is because the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching would not necessarily be seen as a foreign agent in the body. The POG has a preferred molecular weight in the same range as PEG. The structure for POG is shown in Knauf et al., 1988, J. Biol. Chem. 263:15064, and a discussion of POG/IL-2 conjugates is found in U.S. Pat. No. 4,766,106, both of which are hereby incorporated by reference in their entireties.
Another drug delivery system for increasing circulatory half-life is the liposome. Methods of preparing liposome delivery systems are discussed in Gabizon et al., Cancer Research (1982) 42:4734; Cafiso, Biochem Biophys Acta (1981) 649:129; and Szoka, Ann Rev Biophys Eng (1980) 9:467. Other drug delivery systems are known in the art and are described in, e.g., Poznansky et al., Drug delivery systems (R. L. Juliano, ed., Oxford, N.Y. 1980), pp. 253-315; M. L. Poznansky, Pharm Revs (1984) 36:277.
After the liquid pharmaceutical composition is prepared, it is preferably lyophilized to prevent degradation and to preserve sterility. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer""s solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is preferably administered to subjects using those methods that are known to those skilled in the art.
As stated above, the immunotoxins and compositions of this invention are used to treat human patients. The preferred route of administration is parenterally. In parenteral administration, the compositions of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic and nontherapeutic. Examples of such vehicles are saline, Ringer""s solution, dextrose solution, and Hanks+ solution. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. A preferred vehicle is 5% dextrose in saline. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.
The dosage and mode of administration will depend on the individual. Generally, the compositions are administered so that antibodies are given at a dose between 1 :g/kg and 20 mg/kg, more preferably between 20 :g/kg and 10 mg/kg. Preferably, it is given as a bolus dose. Continuous infusion may also be used, if so, the antibodies may be infused at a dose between 1 and 100 :g/kg/min.
The compositions containing the present pharmaceutical compositions or a cocktail thereof (i.e., with other pharmaceutically active proteins) can be administered for therapeutic treatments. In therapeutic applications, compositions are administered to a patient suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate to accomplish this is defined as a xe2x80x9ctherapeutically effective dosexe2x80x9d. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient""s health.
Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the proteins of this invention to effectively treat the patient.